The detection of target molecules and molecular components of larger structures is important in biological and biochemical sciences. The identification, analysis and monitoring of target biochemical or biological analytes, for example, is important for biomedical applications. Current diagnostics and assays employ a variety of methods to detect and analyze target molecules or molecular components (“analytes”) in various environments, both in vitro and in vivo. Certain detection and analysis methods employ fluorescence phenomena. For example, immunoassays often employ antibodies labeled with fluorescent dye molecules (e.g., fluorescein derivatives) to target and detect certain analytes that specifically interact with the antibody. In these methods, a fluorescence signal produced by the fluorescent dye molecule attached to the antibody correlates with antibody-analyte interaction.
In other methods, a fluorescence signal may be altered by interaction between an analyte and a biosensor. Biosensor methods are capable of detecting the activity of analytes such as enzymes. For example, biosensors based on fluorogenic protease substrates comprising casein conjugates of two boron-dipyrromethene (BODIPY) dyes have been shown to be capable of detecting protease activity. This type of biosensor is disclosed in Jones et al., Analytical Biochem. 251, 144-152 (1997). In another example, biosensors based on fluorescence resonance energy transfer (“FRET”) have been developed to detect kinase activity. A biosensor of this type includes a chimeric protein comprising a cyan fluorescent protein and a yellow fluorescent protein, which undergoes a conformational shift in response to phosphorylation. The conformational shift in the protein alters the orientation between the two fluorescent proteins and generates a FRET change. This type of biosensor is disclosed in Zhang et al., Proc. Natl. Acad. Sci. USA 98, 14997-15002, 2001.